Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>β-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with β-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the β-catenin/TCF transcription factor complex.</p> <p>Results</p> <p>We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the β-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and β-catenin. Furthermore, endogenous Tiam1 associated with endogenous β-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.</p> <p>Conclusion</p> <p>Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active β-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.</p

Discovery of Novel Hypermethylated Genes in Prostate Cancer Using Genomic CpG Island Microarrays

BACKGROUND: Promoter and 5' end methylation regulation of tumour suppressor genes is a common feature of many cancers. Such occurrences often lead to the silencing of these key genes and thus they may contribute to the development of cancer, including prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. Using computational and gene-specific validation approaches we have identified a large number of potential epigenetic biomarkers of prostate cancer. Further validation of candidate genes on a separate cohort of low and high grade prostate cancers by quantitative MethyLight analysis has allowed us to confirm DNA hypermethylation of HOXD3 and BMP7, two genes that may play a role in the development of high grade tumours. We also show that promoter hypermethylation is responsible for downregulated expression of these genes in the DU-145 PCa cell line. CONCLUSIONS/SIGNIFICANCE: This study identifies novel epigenetic biomarkers of prostate cancer and prostate cancer progression, and provides a global assessment of DNA methylation in prostate cancer

Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer

Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2’–deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients

Epigenome-wide DNA methylation profiling identifies differential methylation biomarkers in high-grade bladder cancer

Epigenetic changes, including CpG island hypermethylation, occur frequently in bladder cancer (BC) and may be exploited for BC detection and distinction between high-grade (HG) and low-grade (LG) disease. Genome-wide methylation analysis was performed using Agilent Human CpG Island Microarrays to determine epigenetic differences between LG and HG cases. Pathway enrichment analysis and functional annotation determined that the most frequently methylated pathways in HG BC were enriched for anterior/posterior pattern specification, embryonic skeletal system development, neuron fate commitment, DNA binding, and transcription factor activity. We identified 990 probes comprising a 32-gene panel that completely distinguished LG from HG based on methylation. Selected genes from this panel, EOMES, GP5, PAX6, TCF4, and ZSCAN12, were selected for quantitative polymerase chain reaction-based validation by MethyLight in an independent series (n=84) of normal bladder samples and LG and HG cases. GP5 and ZSCAN12, two novel methylated genes in BC, were significantly hypermethylated in HG versus LG BC (P≤.03). We validated our data in a second independent cohort of LG and HG BC cases (n=42) from The Cancer Genome Atlas (TCGA). Probes representing our 32-gene panel were significantly differentially methylated in LG versus HG tumors (P≤.04). These results indicate the ability to distinguish normal tissue from cancer, as well as LG from HG, based on methylation and reveal important pathways dysregulated in HG BC. Our findings were corroborated using publicly available data sets from TCGA. Ultimately, the creation of a methylation panel, including GP5 and ZSCAN12, able to distinguish between disease phenotypes will improve disease management and patient outcomes

EpiTYPER analysis of <i>HOXD3</i>.

<p>(A) PP3 cases 4 and 8 and (B) PP4 cases F and I. Reference lymphocyte is shown for each. Coloured bars represent the average methylation over three replicates with standard error bars displayed.</p

EpiTYPER analysis of <i>BMP7</i>.

<p>(A) PP3 cases 3 and 4 and (B) PP4 cases B and I. Reference lymphocyte is shown for each. Coloured bars represent the average methylation over three replicates with standard error bars displayed.</p

Representative genes and average PGS fold change (across multiple probes) from the top 100 for cancer/reference and progression dataset.

<p>Representative genes and average PGS fold change (across multiple probes) from the top 100 for cancer/reference and progression dataset.</p

Representative homeobox genes showing methylation for cancer/reference and progression dataset (genes in bold overlap with Table 1).

<p>Representative homeobox genes showing methylation for cancer/reference and progression dataset (genes in bold overlap with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004830#pone-0004830-t001" target="_blank">Table 1</a>).</p